NEW ONCOLYTIC NEWCASTLE DISEASE VIRUSES AND RECOMBINANT NDV STRAINS

Abstract

The invention relates to a novel Newcastle Disease Viruses (NDV) and transgene expressing Newcastle Disease Viruses (NDV), which have been demonstrated to possess significant oncolytic activity against mammalian cancers and an improved safety profile. The invention provides novel oncolytic viruses through the use of genetic engineering, including the transfer of foreign genes or parts thereof. The present invention also provides nucleic acids encoding a reverse genetically engineered (rg-)NDV comprising one or more of these foreign genes and having a mutation in the HN gene, said mutation allowing replication of said rgNDV in a cancer cell to a higher level than replication of an otherwise identical rgNDV not having said mutation in the HN gene, as well as a mutation in the F gene, said mutation resulting in a reduced ICPI value as compared to an otherwise identical rgNDV not having said at least one mutation in the F gene.

Claims

1. A Newcastle Disease Virus (NDV), derived from NDV strain MTH-68/H according to SEQ ID No. 1, comprising a viral genome comprising a nucleic acid comprising a nucleic acid sequence encoding a hemagglutinin-neuramidase protein (HN protein) in which phenylalanine (F) at position 277 is substituted to leucine (L), and a nucleic acid sequence encoding a fusion protein (F protein) in which phenylalanine (F) is substituted to serine (S) at position 117 of the F protein, phenylalanine (F) is substituted to leucine (L) at position 190 of the F protein, and leucine (L) is substituted to alanine (A) at position 289 of the F protein.

2. The NDV according to claim 1, wherein the viral genome further comprises a nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M protein) in which glycine (G) is substituted to tryptophane (W) at position 165 of the M protein.

3. The NDV according to claim 1, wherein the viral genome further comprises a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L protein) in which valine (V) is substituted to isoleucine (I) at position 757 of the L protein, and/or phenylalanine (F) is substituted to serine (S) at position 1551 of the L protein, and/or arginine (R) is substituted to leucine (L) at position 1700 of the L protein.

4. The NDV according to claim 3, wherein in the large polymerase protein (L protein) encoded by the nucleic acid sequence tyrosine (Y) is substituted to histidine (H) at position 1717 of the L protein, and/or glutamic acid (E) is substituted to lysine (K) at position 1910 of the L protein.

5. The NDV according to claim 1, wherein the nucleic acid is at least 70% identical to a nucleic acid sequence according to SEQ ID No. 1 to SEQ ID No. 3 of the sequence listing, wherein the sequence identity is determined after best alignment of the sequence of interest with the respective reference sequence.

6. The NDV according to claim 1, wherein the HN protein comprises or consists of the amino acid sequence according to SEQ ID No. 32 of the sequence listing, and/or wherein the F protein comprises or consists of the amino acid sequence according to SEQ ID No. 33 of the sequence listing, and/or wherein the nucleic acid comprises a nucleic acid sequence encoding a membrane (M) protein which M protein comprises or consists of the amino acid sequence according to SEQ ID No. 34 of the sequence listing, and/or wherein the nucleic acid comprises a nucleic acid sequence encoding a large polymerase protein which L protein-comprises or consists of the amino acid sequence according to SEQ ID No. 35 of the sequence listing.

7. The NDV according to claim 1, wherein the NDV is a recombinant virus comprising a nucleic acid sequence encoding at least one foreign gene, the at least one foreign gene being selected from the group consisting of: a gene encoding Atezolizumab, a variant of Atezolizumab or a variant of an antigen-binding part of Atezolizumab, a gene encoding Bevacizumab, an antigen-binding part of Bevacizumab, a variant of Bevacizumab or a variant of an antigen-binding part of Bevacizumab, a gene encoding Lirilumab, an antigen-binding part of Lirilumab, a variant of Lirilumab or a variant of an antigen-binding part of Lirilumab, a gene encoding Relatlimab, an antigen-binding part of Relatlimab, a variant of Relatlimab or a variant of an antigen-binding part of Relatlimab, a gene encoding a gene encoding Monalizumab, an antigen-binding part of Monalizumab, a variant of Monalizumab or a variant of an antigen-binding part of Monalizumab, a gene encoding TRX518, an antigen-binding part of TRX518, a variant of TRX518 or a variant of an antigen-binding part of TRX518, a gene encoding BMS 986178, an antigen-binding part of BMS 986178, a variant of BMS 986178 or a variant of an antigen-binding part of BMS 986178, a gene encoding the protein CD40 (cluster of differentiation 40), a part of CD40, a variant of CD40 or a variant of a part of CD40, a gene encoding the protein CD80, a part of CD80, a variant of CD80 or a variant of a part of CD80, a gene encoding non-secreting human interleukin 12 t(ns)hIL-12), a part of (ns)hIL-12, a variant of (ns)hIL-12 or a variant of a part of (ns)hIL-12, a gene encoding a green fluorescent protein or a part of a green fluorescent protein, a gene encoding Nivolumab, an antigen-binding part of Nivolumab, a variant of Nivolumab or a variant of an antigen-binding part of Nivolumab, a gene encoding Ipilimumab, an antigen-binding part of Ipilimumab, a variant of Ipilimumab or a variant of an antigen-binding part of Ipilimumab, a gene encoding interleukin-12 (IL-12), a part of interleukin-12, a variant of interleukin-12 or a variant of a part of interleukin-12, a gene encoding the non-structural protein NS1 of influenza A virus, a part of the non-structural protein NS1 of influenza A virus, a variant of the non-structural protein NS1 of influenza A virus or a variant of a part of the non-structural protein NS1 of influenza A virus, a gene encoding Apoptin, a part of Apoptin, a variant of Apoptin or a variant of a part of Apoptin, a gene encoding the viral protein B18R from vaccinia virus, a part of the viral protein B18R from vaccinia virus, a variant of the viral protein B18R from vaccinia virus or a variant of a part of the viral protein B18R from vaccinia virus, a gene encoding Theralizumab, an antigen-binding part of Theralizumab, a variant of Theralizumab or a variant of an antigen-binding part of Theralizumab, a gene encoding an antibody, directed to CD40 or an antigen-binding part directed to CD40 (anti-CD40), a gene encoding an antibody, directed to CD80 or an antigen-binding part directed to CD80 (anti-CD80), a gene encoding an antibody directed to CA 15-3 or an antigen-binding part directed to CA 15-3 (anti-CA 15-3), according to SEQ. ID. No. 22 of the sequence listing, a gene encoding an antibody directed to CA 19-9 or an antigen-binding part directed to CA 19-9 (anti-CA 19-9), according to SEQ. ID. No. 23, a gene encoding Sofituzumab, an antigen-binding part of Sofituzumab, a variant of Sofituzumab or a variant of an antigen-binding part of Sofituzumab, a gene encoding Cetuximab, an antigen-binding part of Cetuximab, a variant of Cetuximab or a variant of an antigen-binding part of Cetuximab, a gene encoding Trastuzumab, an antigen-binding part of Trastuzumab, a variant of Trastuzumab or a variant of an antigen-binding part of Trastuzumab, a gene encoding the antibody BIL=3s, an antigen-binding part of BIL=3 s, a variant of BIL=3 s or a variant of an antigen-binding part of BIL=3s, a gene encoding the antibody J591, an antigen-binding part of J591, a variant of J591 or a variant of an antigen-binding part of J591, a gene encoding Ramucirumab, an antigen-binding part of Ramucirumab, a variant of Ramucirumab or a variant of an antigen-binding part of Ramucirumab, a gene encoding the protein TRAIL, a part of TRAIL, a variant of TRAIL or a variant of a part of TRAIL, and any combination of these genes, parts or variants.

8. A nucleic acid comprising a nucleic acid sequence encoding a Newcastle Disease Virus (NDV) according to claim 1, the nucleic acid comprising the nucleic acid sequence encoding the hemagglutinin-neuramidase (HN) protein, where the nucleic acid sequence encodes HN.sup.F277L, and the nucleic acid sequence encoding a fusion (F) protein, where the nucleic acid sequence encodes F.sup.F117S, F.sup.F190L, and F.sup.L289A.

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. A recombinant Newcastle Disease Virus (NDV), derived from NDV strain MTH-68/H according to SEQ ID No. 1, wherein the recombinant NDV comprises a viral genome comprising a nucleic acid comprising a nucleic acid sequence encoding at least one foreign gene, the at least one foreign gene being selected from the group consisting of: a gene encoding an antibody directed to protein PD-1 or an antigen-binding part directed to protein PD-1 (anti-PD-1), a gene encoding an antibody directed to the surface protein CTLA-4 or an antigen-binding part directed to protein CTLA-4 (anti-CTLA-4), a gene encoding a protein which improves the cellular immune response and the ability of T cells to enter tumor cells, or a part thereof which improves the cellular immune response and the ability of T cells to enter tumor cells, a gene encoding a protein with the ability to modulate the virus replication cycle, or a part thereof with the ability to modulate the virus replication cycle, a gene encoding a protein with the ability to selectively induce apoptosis in human tumor cells, but not in normal human cells, or a part thereof with the ability to selectively induce apoptosis in human tumor cells, but not in normal human cells, a gene encoding a protein that reduces or inhibits IFN expression, or a part thereof that reduces or inhibits IFN expression a gene encoding the protein CD40 (cluster of differentiation 40), a part of CD40, a variant of CD40 or a variant of a part of CD40, a gene encoding an antibody directed to CD28 or an antigen-binding part directed to CD28 (anti-CD28), a gene encoding an antibody directed to CD40 or an antigen-binding part directed to CD40 (anti-CD40), a gene encoding an antibody directed to CD80 or an antigen-binding part directed to CD80 (anti-CD80), a gene encoding an antibody directed to CA 15-3 or an antigen-binding part directed to CA 15-3 (anti-CA 15-3), a gene encoding an antibody directed to CA 19-9 or an antigen-binding part directed to CA 19-9 (anti-CA 19-9), a gene encoding an antibody directed to CA 125 or an antigen-binding part directed to CA 125 (anti-CA 125), a gene encoding an antibody directed to EGFR or an antigen-binding part directed to EGFR (anti-EGFR), a gene encoding an antibody directed to HER2 or an antigen-binding part directed to HER2 (anti-HER2), a gene encoding an antibody directed to nfP2X.sub.7 or an antigen-binding part directed to nfP2X.sub.7 (anti-nfP2X.sub.7), a gene encoding an antibody directed to PSMA or an antigen-binding part directed to PSMA (anti-PSMA), a gene encoding an antibody, directed to VEGFR or an antigen-binding part directed to VEGFR (anti-VEGFR), a gene encoding a protein that induces the process of apoptosis by binding to a death receptor or a part thereof that induces the process of apoptosis by binding to a death receptor, and any combination of these genes, parts or variants, wherein the viral genome comprises a nucleic acid sequence encoding a hemagglutinin-neuramidase protein (HN protein) in which phenylalanine (F) is substituted to leucine (L) at position 277, and a nucleic acid sequence encoding a fusion protein (F protein) in which phenylalanine (F) is substituted to serine (S) at position 117 of the F protein, and phenylalanine (F) is substituted to leucine (L) at position 190 of the F protein.

15. The recombinant NDV according to claim 14, wherein the viral genome comprises a nucleic acid sequence encoding a matrix protein (M protein) in which glycine (G) is substituted to tryptophane (W) at position 165 of the M protein.

16. The recombinant NDV according to claim 14, wherein the viral genome comprises a nucleic acid sequence encoding a large polymerase protein (L protein) in which valine (V) is substituted to isoleucine (I) at position 757 of the L protein, and/or phenylalanine (F) is substituted to serine (S) at position 1551 of the L protein, and/or arginine (R) is substituted to leucine (L) at position 1700 of the L protein.

17. The recombinant NDV according to claim 16, wherein in the large polymerase protein (L protein) encoded by the nucleic acid sequence tyrosine (Y) is substituted to histidine (H) at position 1717 of the L protein, and/or glutamic acid (E) is substituted to lysine (K) at position 1910 of the L protein.

18. The recombinant NDV according to claim 14, wherein the nucleic acid is at least 70% identical to a nucleic acid sequence according to SEQ ID No. 1, SEQ ID No. 36, SEQ ID No. 37 or SEQ ID No. 38 of the sequence listing, wherein the sequence identity is determined after best alignment of the sequence of interest with the respective reference sequence.

19. The recombinant NDV according to claim 14, wherein the HN protein comprises or consists of the amino acid sequence according to SEQ ID No. 32 of the sequence listing, and/or wherein the F protein comprises or consists of the amino acid sequence according to SEQ ID No. 39 of the sequence listing, and/or wherein the viral genome comprises a nucleic acid sequence encoding a matrix (M) protein, which M protein comprises or consists of the amino acid sequence according to SEQ ID No. 34 of the sequence listing, and/or wherein the viral genome comprises a nucleic acid sequence encoding a large polymerase (L) protein, which L protein comprises or consists of the amino acid sequence according to SEQ ID No. 35 of the sequence listing.

20. A nucleic acid encoding the recombinant Newcastle Disease Virus (NDV) according to claim 14, the nucleic acid comprising a transgenic construct, wherein said transgenic construct comprises a nucleic acid sequence encoding at least one protein selected from the group consisting of: an antibody directed to protein PD-1 or an antigen-binding part directed to protein PD-1 (anti-PD-1), an antibody directed to the surface protein CTLA-4 or an antigen-binding part directed to protein CTLA-4 (anti-CTLA-4), a protein which improves the cellular immune response and the ability of T cells to enter tumor cells, or a part thereof which improves the cellular immune response and the ability of T cells to enter tumor cells, a protein with the ability to modulate the virus replication cycle, or a part thereof with the ability to modulate the virus replication cycle, a protein with the ability to selectively induce apoptosis in human tumor cells, but not in normal human cells, or a part thereof with the ability to selectively induce apoptosis in human tumor cells, but not in normal human cells, a protein that reduces or inhibits IFN expression such as an IFN-beta receptor, or a part thereof that reduces or inhibits IFN expression such as an IFN-beta receptor, a protein CD40 (cluster of differentiation 40), a part of CD40, a variant of CD40 or a variant of a part of CD40, an antibody, directed to CD28 or an antigen-binding part directed to CD28 (anti-CD28), an antibody, directed to CD40 or an antigen-binding part directed to CD40 (anti-CD40), an antibody, directed to CD80 or an antigen-binding part directed to CD80 (anti-CD80), an antibody directed to CA 15-3 or an antigen-binding part directed to CA 15-3 (anti-CA 15-3), an antibody directed to CA 19-9 or an antigen-binding part directed to CA 19-9 (anti-CA 19-9), an antibody directed to CA 125 or an antigen-binding part directed to CA 125 (anti-CA 125), an antibody, directed to EGFR or an antigen-binding part directed to EGFR (anti-EGFR), an antibody, directed to HER2 or an antigen-binding part directed to HER2 (anti-HER2), an antibody directed to nfP2X.sub.7 or an antigen-binding part directed to nfP2X.sub.7 (anti-nfP2X.sub.7), an antibody directed to PSMA or an antigen-binding part directed to PSMA (anti-PSMA), an antibody, directed to VEGFR or an antigen-binding part directed to VEGFR (anti-VEGFR), a protein that induces the process of apoptosis by binding to a death receptor, or a part thereof that induces the process of apoptosis by binding to a death receptor, any combination of these proteins, parts or variants, wherein the nucleic acid in addition comprises the nucleic acid sequence encoding the hemagglutinin-neuramidase protein (HN protein) with an amino acid substitution at position 277 to an amino acid with a hydrophobic side chain other than phenylalanine, namely HN.sup.F277L, and the nucleic acid sequence encoding the fusion protein (F protein) with an amino acid substitution at position 117 to an amino acid with a hydroxylated side chain, namely F.sup.F117S, and with an amino acid substitution at position 190 to an amino acid with an aliphatic side chain, namely F.sup.F190L.

21. The nucleic acid according to claim 20, further comprising a nucleic acid sequence encoding a matrix protein (M protein) with an amino acid substitution at position 165 to an amino acid with an aromatic side chain, namely M.sup.G165W.

22. (canceled)

23. (canceled)

24. The nucleic acid according to claim 20, wherein the nucleic acid comprises at least one of the nucleic acids according to SEQ ID No. 1, SEQ ID No. 36, SEQ ID No. 37 or SEQ ID No. 38 of the sequence listing or variants thereof, the variants having a sequence identity of at least 75%, to the respective sequence, wherein the sequence identity is determined after best alignment of the sequence of interest with the respective reference sequence.

25. The NDV according to claim 7, wherein the nucleic acid comprising the nucleic acid sequence encoding the at least one foreign gene comprises or is a nucleic acid sequence according to any one the nucleic acid sequences according to SEQ ID No. 5 to 30 of the sequence listing.

26. (canceled)

27. (canceled)

28. A method of treating cancer in a subject considered in need thereof, the method comprising administering the NDV according to claim 1 to the subject, wherein the cancer is selected from the group consisting of brain tumors, bone tumors, soft tissue tumors, gynecological tumors, gastrointestinal tumors, pancreas tumors, prostate tumors, lung tumors, ear tumors, nose tumors, throat tumors, tongue tumors, and skin tumors.

29-31. (canceled)

32. The recombinant NDV according to claim 14, wherein the at least one foreign gene is selected from the group consisting of: the gene encoding an antibody directed to protein PD-1 or an antigen-binding part directed to protein PD-1 (anti-PD-1), which gene encodes Nivolumab, an antigen-binding part of Nivolumab, a variant of Nivolumab or a variant of an antigen-binding part of Nivolumab, the gene encoding an antibody directed to the surface protein CTLA-4 or an antigen-binding part directed to protein CTLA-4 (anti-CTLA-4), which gene encodes Ipilimumab, an antigen-binding part of Ipilimumab, a variant of Ipilimumab or a variant of an antigen-binding part of Ipilimumab, the gene encoding a protein which improves the cellular immune response and the ability of T cells to enter tumor cells, or a part thereof which improves the cellular immune response and the ability of T cells to enter tumor cells, which gene encodes interleukin-12 (IL-12), a part of interleukin-12, a variant of interleukin-12 or a variant of a part of interleukin-12, the gene encoding a protein with the ability to modulate the virus replication cycle, or a part thereof with the ability to modulate the virus replication cycle, which gene encodes the non-structural protein NS1 of influenza A virus, a part of the non-structural protein NS1 of influenza A virus, a variant of the non-structural protein NS1 of influenza A virus or a variant of a part of the non-structural protein NS1 of influenza A virus, the gene encoding a protein with the ability to selectively induce apoptosis in human tumor cells, but not in normal human cells, or a part thereof with the ability to selectively induce apoptosis in human tumor cells, but not in normal human cells, which gene encodes Apoptin, a part of Apoptin, a variant of Apoptin or a variant of a part of Apoptin, the gene encoding a protein that reduces or inhibits IFN expression, or a part thereof that reduces or inhibits IFN expression, which gene encodes the viral protein B18R from vaccinia virus, a part of the viral protein B18R from vaccinia virus, a variant of the viral protein B18R from vaccinia virus or a variant of a part of the viral protein B18R from vaccinia virus, the gene encoding the protein CD40 (cluster of differentiation 40), a part of CD40, a variant of CD40 or a variant of a part of CD40, the gene encoding an antibody directed to CD28 or an antigen-binding part directed to CD28 (anti-CD28), which gene encodes Theralizumab, an antigen-binding part of Theralizumab, a variant of Theralizumab or a variant of an antigen-binding part of Theralizumab, the gene encoding an antibody directed to CA 15-3 or an antigen-binding part directed to CA 15-3 (anti-CA 15-3), having a sequence which comprises or is the nucleic acid sequence SEQ. ID. No. 22 of the sequence listing, a gene encoding an antibody directed to CA 19-9 or an antigen-binding part directed to CA 19-9 (anti-CA 19-9), having a sequence which comprises or is the nucleic acid sequence SEQ. ID. No. 23 of the sequence listing, the gene encoding an antibody directed to CA 125 or an antigen-binding part directed to CA 125 (anti-CA 125), which gene encodes Sofituzumab, an antigen-binding part of Sofituzumab, a variant of Sofituzumab or a variant of an antigen-binding part of Sofituzumab, the gene encoding an antibody directed to EGFR or an antigen-binding part directed to EGFR (anti-EGFR), which gene encodes Cetuximab, an antigen-binding part of Cetuximab, a variant of Cetuximab or a variant of an antigen-binding part of Cetuximab, the gene encoding an antibody directed to HER2 or an antigen-binding part directed to HER2 (anti-HER2), which gene encodes Trastuzumab, an antigen-binding part of Trastuzumab, a variant of Trastuzumab or a variant of an antigen-binding part of Trastuzumab, the gene encoding an antibody directed to nfP2X.sub.7 or an antigen-binding part directed to nfP2X.sub.7 (anti-nfP2X.sub.7), which gene encodes BIL=3s, an antigen-binding part of BIL=3 s, a variant of BIL=3s or a variant of an antigen-binding part of BIL=3s, the gene encoding an antibody directed to PSMA or an antigen-binding part directed to PSMA (anti-PSMA), which gene encodes J591, an antigen-binding part of J591, a variant of J591 or a variant of an antigen-binding part of J591, the gene encoding an antibody directed to VEGFR or an antigen-binding part directed to VEGFR (anti-VEGFR), which gene encodes Ramucirumab, an antigen-binding part of Ramucirumab, a variant of Ramucirumab or a variant of an antigen-binding part of Ramucirumab, the gene encoding a protein that induces the process of apoptosis by binding to a death receptor or a part thereof that induces the process of apoptosis by binding to a death receptor which gene encodes TRAIL, a part of TRAIL, a variant of TRAIL or a variant of a part of TRAIL and any combination of these genes, parts or variants.

Description

FIGURES

[0481] FIG. 1 is a schematic presentation of the NDV reverse genetics system. The upper part shows the composition of the full-length cDNA plasmid which contains the full-length NDV cDNA (encoding the nucleocapsid protein (NP), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin-neuraminidase (HN), and the RNA-dependent RNA polymerase (L)) cloned behind the bacteriophage T7 RNA Polymerase promoter (T7P; yellow triangle) and followed by a ribozyme sequence (Rz) and T7 transcription termination signal (T7T).

[0482] A suitable host cell (shaded round-cornered box) is infected with a recombinant Fowlpox virus that expresses T7 DNA-dependent RNA polymerase (Fowlpox-T7) and subsequently co-transfected with the full-length cDNA plasmid and three helper plasmids containing the genes encoding the NDV NP, P and L proteins, respectively. Transcription of the full-length cDNA results in the generation of the NDV antigenome RNA which is encapsidated by NP protein then transcribed and replicated by the RNA-leading to the generation of infectious NDV.

[0483] FIG. 2 is a schematic presentation of the genome of the rgNDV-mutants according to the present invention. The figure shows the position of the respective foreign gene which is inserted as extra transcription unit into the rgNDV-mutant genome between the P and M genes.

[0484] FIG. 3 shows growth kinetics in HeLa cells. HeLa cells were infected at a multiplicity of infection (MOI) of 0.01 with the indicated viruses. At 0 h, 8 h, 24 h and 48 h after infection, the amount of infectious virus in the supernatant was determined by end-point titration on QM5 cells. MTH68: NDV strain MTH-68/H; MutHu: NDV-Mut HN(F277L)/M(G165W); rgMTH68: NDV strain MTH-68/H derived by reverse genetics from cloned full-length cDNA; rgMutHu: NDV strain NDV-Mut HN(F277L)/M(G165W) derived by reverse genetics from cloned full-length cDNA; rgMutHu(HNL277F): rgMutHu in which the amino acid mutation at position 277 in the HN gene was converted from L back to F; rgMutHu(MW165G): rgMutHu in which the amino acid mutation at position 165 in the M gene was converted from W back to G.

[0485] FIG. 4 shows the expression of CD80 in a recombinant Nothabene-2-strain. FIG. 4a shows the recombinant strain, FIG. 4b is the negative control. For detection an anti-CD80 antibody was used.

[0486] FIG. 5 shows syncytium formation due to the F(L289A) mutation. FIG. 5a is rgNothabene-2-F2L3+F(L289A) (i.e. F3L3) according to the present invention, FIG. 5b is rgNothabene-2-F2L3.

EXAMPLES

Example 1. Nucleotide Sequence Analysis of Mutant NDV-Mut HN(F277L)/M(G165W)

[0487] We identified a spontaneous mutant of an oncolytic NDV strain MTH-68/H (Csatary et al., 1999, Anticancer Res. 19:635-638; further called MTH68). The replication capacity of the mutant strain (designated NDV-Mut HN(F277L)/M(G165W) in a variety of human neoplastic cell lines, as well as autologous primary tumors, is greatly enhanced as compared to the original MTH-68/H strain (also referred to as MTH68 strain). We analyzed its nucleotide sequence and found that, compared to MTH68, NDV-HN(F277L)/M(G165W) has two nucleotide mutations, one leading to an amino acid substitution in the M protein (G165W) and the other in the HN protein (F277L).

Example 2. A Reverse Genetics System that Allows Genetic Modification of NDV-Strains

[0488] 2.1 Reverse Genetics

[0489] In order to be able to genetically modify the genome of an RNA virus such as NDV, a manipulatable genetic system must be developed that uses a copy of the full viral RNA (vRNA) genome in the form of DNA. This full-length cDNA is amenable to genetic modification by using recombinant DNA techniques. The authentic or modified cDNA can be converted back into vRNA in cells, which in the presence of the viral replication proteins results in the production of a new modified infectious virus. Such ‘reverse genetics systems’ have been developed in the last few decades for different classes of RNA viruses. This system enables the rapid and facile introduction of mutations and deletions and the insertion of a transgene transcriptional unit, thereby enabling the changing of the biological properties of the virus.

[0490] Reverse genetics systems for several NDV strains, including lentogenic as well as velogenic strains, were developed by the Central Veterinary Institute (CVI), part of Wageningen University and Research, currently Wageningen Bioveterinary Research (WBVR) under the supervision of Dr. Ben Peeters (Peeters et al., 1999, J. Virol. 73:5001-9; de Leeuw et al., 2005, J. Gen. Virol. 86:1759-69; Dortmans et al., 2009, J. Gen. Virol. 90:2746-50). In order to generate a reverse genetics system for providing the NDV nucleic acids and strains according to the present invention, a similar approach was used. Details of the procedure can be found in the above cited papers and in the paragraphs below. Briefly, the system consists of 4 components, i.e., a transcription plasmid containing the full-length (either authentic or genetically modified) cDNA of the virus, which is used to generate the vRNA, and 3 expression plasmids (‘helper plasmids’) containing the NP, P and L genes of NDV respectively, which are used to generate the vRNA-replication complex (consisting of NP, P and L proteins). Transcription of the cDNA (i.e. conversion of the cDNA into vRNA) and expression of the NP, P and L genes by the helper plasmids is driven by a T7 promoter. The corresponding T7 DNA-dependent RNA polymerase (T7-RNAPol) is provided by a helper-virus (Fowlpox-T7).

[0491] In order to rescue virus, the 4 plasmids are co-transfected into Fowlpox-T7 infected cells (FIG. 1). Three to five days after transfection, the supernatant is inoculated into specific-pathogen-free embryonated chicken eggs (ECE) and incubated for 3 days. Infectious virus that is produced by transfected cells will replicate in the ECE and progeny virus can be harvested from the allantois fluid.

[0492] In order to develop a reverse genetics system for NDV the following steps were followed: [0493] Generation of sub-genomic NDV and foreign gene cDNA's by RT-PCR [0494] Assembly of full-length cDNA in a transcription vector [0495] Cloning of each of the NP, P and L genes into an expression vector [0496] Verify nucleotide sequence of full-length cDNA and helper-plasmids [0497] Repair nucleotide differences resulting from the cloning procedure, if necessary [0498] Rescue of infectious virus from cDNA using co-transfection (FIG. 1)

[0499] 2.2 Construction of Full-Length NDV-Mut HN(F277L)/M(G165W) cDNA, NDV-Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L) and Helper Plasmids

[0500] NDV-Mut HN(F277L)/M(G165W) and NDV-Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L) (passage 28 HeLa cells) were used for the isolation of vRNA using standard procedures. The vRNA was used to generate first-strand cDNA by means of Reverse Transcriptase followed by PCR to generate 4 sub-genomic cDNA fragments (designated C1, C2, C3 and C8). The full-length cDNA of NDV-MutHu was assembled from these fragments and cloned in the transcription vector pOLTV5 (Peeters et al., 1999, J. Virol. 73:5001-5009) by a combination of In-Fusion® cloning and classical cloning using restriction enzymes. An overview of the procedure is shown in FIG. 2, and further details can be found in Appendix 1 of this reference. The NP, P and L-genes of NDV-MutHu were obtained by RT-PCR (Appendix 1) and cloned in the expression plasmid pCVI which was derived by deletion of a ClaI restriction fragment from pCI-neo (Promega).

[0501] 2.3 Nucleotide Sequence Analysis

[0502] Nucleotide sequence analysis was used to verify that the sequence of pFL-NDV Mut HN(F277L)/M(G165W) and pFL-NDV Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L) were correct. A few nucleotides which differed from the Reference sequence were repaired. Silent mutations (i.e., not leading to an amino acid change) may be left unchanged.

[0503] 2.4 Rescue of Infectious Virus from pFL-NDV Mut HN(F277L)/M(G165W)

[0504] In order to generate infectious virus, we used the co-transfection system described above (and illustrated in FIG. 1) to transfect QM5 cells (derived from Quail) using plasmid pFL-NDV_Mut HN(F277L)/M(G165W) and the helper plasmids pCVI-NP.sup.Mut HN(F277L)/M(G165W), pCVI-P.sup.Mut HN(F277L)/M(G165W) and pCVI-L.sup.Mut HN(F277L)/M(G165W). Rescue of infectious virus was successful as demonstrated by the presence of infectious virus in the allantoic fluid of ECE that were inoculated with the transfection supernatant (data not shown). The identity of the rescued virus was determined by RT-PCR followed by sequencing. The rescued virus was designated rgMut HN(F277L)/M(G165W). NDV-Mut HN(F277L)/M(G165W) and rgMut HN(F277L)/M(G165W) have similar growth kinetics in HeLa cells.

[0505] 2.5 Rescue of Infectious Virus from pFL-NDV Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L)

[0506] In order to generate infectious virus, we used the co-transfection system described above (and illustrated in FIG. 1) to transfect QM5 cells (derived from Quail) using plasmid pFL-NDV Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L) and the helper plasmids pCVI-NP.sup.Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L), pCVI-P.sup.Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L) and pCVI-L.sup.Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L). Rescue of infectious virus was successful as demonstrated by the presence of infectious virus in the allantoic fluid of ECE that were inoculated with the transfection supernatant (data not shown). The identity of the rescued virus was determined by RT-PCR followed by sequencing. The rescued virus was designated rgMut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L).

Example 3. Identify Whether One or Both of the Amino Acid Substitutions in Mut HN(F277L)/M(G165W) are Responsible for the Difference in Growth Kinetics Between Mut HN(F277L)/M(G165W) and the Parent Strain MTH68

[0507] 3.1 Growth Kinetics in HeLa Cells

[0508] The rescued rg-viruses (Table 1) as well as the original Mut HN(F277L)/M(G165W) and MTH68 viruses were used to determine their growth-kinetics in HeLa cells. Briefly, 4×10.sup.6 HeLa cells were seeded in 25 cm.sup.2 cell culture flasks and grown overnight. The cells were infected using a MOI of 0.01 (i.e., 1 infectious virus particle per 100 cells), and at 8, 24 and 48 hours after infection the virus titer in the supernatant was determined by end-point titration on QM5 cells.

[0509] The data (FIG. 3) indicate that strains Mut HN(F277L)/M(G165W) and rgMut HN(F277L)/M(G165W) yield at least 10-fold higher virus titers than MTH68. Furthermore, the data indicate that the mutation at amino acid position 277 in the HN gene is responsible for this effect. The M mutation does not seem to have an effect. This can be best seen when looking at the virus titers 24 h after infection, or even better when comparing the increase in virus titer between 8h and 24h (the exponential growth phase). The virus titer shows an increase of 3.5 (log 10) for Mut HN(F277L)/M(G165W), rgMut HN(F277L)/M(G165W) and rgMut HN(F277L)/M(W165G), whereas this is 2.5 for MTH68, 2.7 for rgMut HN(L277F) and 3.0 for rgMTH68 (Table 3).

[0510] rgMut HN(F277L)/M(W165G) is a strain in which the mutation in the M gene has been restored in accordance with the NDV MTH-68/H.

TABLE-US-00007 TABLE 1 Virus titers (log10 TCID50/ml) Time after infection (h) Virus 0 h 8 h 24 h 48 h MTH68 4.8 4.5 7.0 7.0 Mut HN(F277L)/M(G165W) 5.0 4.3 7.8 8.3 rgMTH68 5.0 4.0 7.0 7.5 rgMut HN(L277F) 5.0 4.3 7.0 7.3 rgMut M(W165G) 4.8 4.3 7.8 7.5 rgMutHu 5.3 4.5 8.0 8.0

Example 4. Generation of Recombinant NDV-Strains with Enhanced Oncolytic and Immune Stimulating Properties Due to the Expression of Different Therapeutic Proteins

[0511] The recombinant NDV-strains expressing one of the foreign genes of the present invention can be generated by means of the previously established reverse genetics system described above. The respective gene is to be inserted into the full-length cDNA of e.g. NDV-Mut HN(F277L)/M(G165W)/F(F117S)/F(F190L)/L(V757I)/L(F1551S)/L(R1700L) between the P and the M genes. To this end the open reading frames of the foreign genes may be fused via a 2A sequence. The genes were provided with the necessary NDV gene-start and gene-end sequences in order to allow transcription by the vRNA polymerase. FIG. 2 shows the final constellation of the recombinant viruses.

[0512] Infectious virus can be rescued, and virus stocks can be prepared by two passages in HeLa cells. Expression of the respective foreign gene can be determined and quantified by using a total human IgG ELISA (Invitrogen).

REFERENCES

[0513] Blach-Olszewska et al., (1977) Why HeLa cells do not produce interferon? Arch Immunol Ther Exp (Warsz) 25:683-91. [0514] Chng et al., (2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, mAbs, 7:2, 403-412; http://dx.doi.org/10.1080/19420862.2015.1008351. [0515] Enoch et al., (1986) Activation of the Human beta-Interferon Gene Requires an Interferon-Inducible Factor, Mol. Cell. Biol. 6:801-10. [0516] Haryadi et al., (2015) Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells. PLoS ONE 10(2): e0116878. doi:10.1371/journal.pone.0116878. [0517] Schirrmacher, (2015) Oncolytic Newcastle disease virus as a prospective anti-cancer therapy. A biologic agent with potential to break therapy resistance. Expert Opin. Biol. Ther. 15:17 57-71 [0518] Zamarin et al., (2014) Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy. Sci. Transl. Med. 6(226). [0519] Zamarin & Palese, (2017) Oncolytic Newcastle Disease Virus for cancer therapy: old challenges and new directions. Future Microbiol. 7: 347-67.

TABLE-US-00008 APPENDIX 1 primers used for the generation of cDNA fragments and helper-plasmids cDNA fragments Sequence Fragment Size Primer (5′-3′) C1 3.6 kb Noss-09 ACGACTCACT (SEQ ID ATAGGaccaa No. 40) acagagaatc cgtgag Noss-121 CCGGGAAGAT (SEQ ID CCAGGgcact No. 41) cttcttgcat gttac C2 3.7 kb Noss-122 GGGCCTGCCT (SEQ ID CACTAtggtg No. 42) gtaacatgca agaag Noss-123 TGCATGTTAC (SEQ ID CACCAatgtg No. 43) tcattgtatc gcttg C3 5.7 kb Noss-125 CAAGAAGGGA (SEQ ID GATACgtaat No. 44) atacaagcga tacaatg Noss-126 TCGCTTGTAT (SEQ ID ATTACttgtt No. 45) gtagcaaaga gcacc C8 2.0 kb Noss-133 GGCCTGGATC (SEQ ID TTCCCattat No. 46) gctgtctgta tacggtgc Noss-10 ATGCCATGCC (SEQ ID GACCCaccaa No. 47) acaaagactt ggtgaatg iPCR 2.5 kb Noss-17 CCTATAGTGA pOLTV5 (SEQ ID GTCGTATTAA (StuI) No. 48) TTTC Noss-128 CCTGGATCTT (SEQ ID CCCGGGTCGG No. 49) iPCR 2.5 kb Noss-137 GGGTCGGCAT pOLTV5 (SEQ ID GGCATCTCCA (Smal) No. 50) CC Noss-138 GGGAAGATCC (SEQ ID AGGCCTATAG No. 51) TG Helper-plasmids (generated by In-Fusion® cloning in pCVI) Gene primer sequence NP Noss-22 CTCTAGAGTC (SEQ ID GACCCttctg No. 52) ccaacatgtc ttccg Koss-23 GGGAAGCGGC (SEQ ID CGCCCgtcgg No. 53) tcagtatccc cagtc Noss-24 CTCTAGAGTC (SEQ ID GACCCcagag No. 54) tgaagatggc caccttc P Noss- GGGAAGCGGC 25n CGCCCgtagt (SEQ ID agtgatcagc No. 55) cattc L Noss-26 CTCTAGAGTC (SEQ ID GACCCgggta No. 56) ggacatggcg ggctc Noss-27 GGGAAGCGGC (SEQ ID CGCCCtgcct No. 57) ttaagagtca cagttac